Summary of Work: The analgesic activity of recently developed opioidmimetic substances was determined in vivo in mice in comparison to morphine and using specific opiate-receptor antagonists to assess their mode of action. The antinociception profile paralleled that of the in vitro functional pharmacological data using guinea-pig ileum and mouse vas deferens assay systems, and reflected the opioid-receptor binding affinity (see Project 1 for details on the interaction of compounds with delta- and mu-opioid receptors). A series of novel Dmt-containing alkyl and those containing a pyrazinone group as opioidmimetic compounds had exceptional activity in vivo. In particular, one of which was 60- to 71-fold more potent than morphine in generating analgesia by the tail-flick test after i.c.v. administration; the spinal (tail-flick test) is more potent than the supraspinal effects (hot-plate test) by a factor of 2-3. In particular, the compound 3-[4'-Dmt-aminobutyl)-6-(3'-Dmt-aminopropyl)-5-methyl-2(1H)pyrazinone had very high affinity (Ki mu = 0.02 nM), selectivity (delta/mu = 1,520) and agonist activity (GPI, IC50 = 1.7 nM) with weak activity toward the delta receptor in all assay systems. This compound acted to produce spinal antinociception primarily through spinal antinociception using the mu-2 receptor subtype; however, the mu-1 subtype dominates supraspinally. The s.c. injection produced CNS-mediated antinociception, providing evidence the compound passed through membrane barriers in both the gastrointestinal epithelium and in the microcapillary tight junctions that regulate the blood-brain barrier (BBB). However, a tolerance was obtained after a week of daily injections that was equivalent to morphine and suggests that both substances act through similar mechanisms at mu-opioid receptors. Another class of Dmt-containing pyrazinone compounds, the 3,6-bis-[Dmt-NH-(CH2)n]-2(1H)-pyrazinone compounds not only exhibited central (CNS) mediated analgesia, but also were orally bioavailable opioid mimetics. These symmetric substances displayed high affinity for mu-opioid receptors (Ki = 0.04-0.12 nM) and potent angonism on GPI (IC50 = 1.3-1.9 nM). They produced analgesia in vivo by i.c.v. administration that was 50- to 63-fold more potent than morphine, but only about half as potent when injected s.c. or administered orally, which is still orders of magnitude greater than other endogenous opioid peptides and, importantly, were unmodified by glycosylation, adamantane, triglycerides, halogens, antibody, biotin, bulky organic molecules, esterification of a C-terminal carboxyl group (of which none exist in these compounds) or O-acylation of the phenolic hydroxyal group of Tyr or Dmt, nor was it necessary to absorb them onto polysorbate coated nanoparticles to induced uptake through the BBB.